Oxyntomodulin for Preventing or Treating Excess Weight

ABSTRACT

Compositions and methods for use in the prevention or treatment of excess weight in a mammal have been developed. The compositions comprise oxyntomodulin which is shown to reduce food intake.

The present application is a continuation application of U.S.application Ser. No. 10/488,341, filed Nov. 8, 2004 (allowed), which isthe U.S. National Phase of International Application No. PCT/GB02/04082,filed Sep. 9, 2002, which claims the benefit of British priorityapplication no. UK 0121709.0, filed Sep. 7, 2001.

The present invention relates to compositions and methods for use inweight loss in mammalian animals.

One of the diseases with the highest incidence but which lacks effectivetreatment is obesity. It is a debilitating condition which reducesquality of life and substantially increases the risk of other diseases.

In the USA 25% of the adult population is now considered to beclinically obese. It has been estimated that $45 billion of UShealthcare costs, or 8% per annum of total healthcare spending, is adirect result of obesity. In Europe the problem is increasing. It hasbeen predicted that without new approaches over 20% of the UK populationwill be clinically obese by 2005. The fact that obesity is a metabolicdisease is being increasingly recognised by the medical profession andthe health authorities. There is, however, a shortage of effective andsafe drugs which can be used in conjunction with diet and exercise forthe long-term management of obesity.

It is an object of the present invention to provide such drugs and alsoto provide means to identify and develop such drugs.

Preproglucagon is a 160 amino acid polypeptide which is cleaved in atissue specific manner by prohormone convertase-1 and -2 giving rise toa number of products with a variety of functions in both the centralnervous system (CNS) and peripheral tissues. In the intestine and in theCNS, the major post-translational products of preproglucagon cleavageare glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2),glicentin and oxyntomodulin (OXM), as shown in Figure A. To date, norole in the CNS has been demonstrated for OXM.

While GLP-1 and GLP-2 have been shown to inhibit food intake, no suchrole has been demonstrated for the distinct peptide OXM. The importanceof OXM as a biologically active peptide has not been demonstrated.

It has been surprisingly found that contrary to expectations, the OXMpeptide can inhibit food intake and reduce weight.

Accordingly, the present invention provides, according to a firstaspect, a composition comprising OXM, for use in the prevention ortreatment of excess weight in a mammal.

In this text, the term “oxyntomodulin” is the same as “OXM” and relatesto any composition which includes an OXM peptide sequence or an analoguethereof as follows:

OXM sequences are well known and documented in the art. The presentinvention relates to all of the sequences recited herein including, inparticular, the OXM human sequence (which is the same as the rat,hamster and bovine OXM sequence), as follows:

(SEQ ID NO: 4) His Ser Gln Gly Thr Phe Thr Ser Asp TyrSer Lys Tyr Leu Asp Ser Arg Arg Ala GlnAsp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn Arg Asn Asn Ile Ala,the OXM angler fish sequence as follows:

(SEQ ID NO: 2) His Ser Glu Gly Thr Phe Ser Asn Asp TyrSer Lys Tyr Leu Glu Asp Arg Lys Ala GlnGlu Phe Val Arg Trp Leu Met Asn Asn Lys Arg Ser Gly Val Ala Glu,and the eel OXM sequence as follows:

(SEQ ID NO: 3) His Ser Gln Gly Thr Phe Thr Asn Asp TyrSer Lys Tyr Leu Glu Thr Arg Arg Ala GlnAsp Phe Val Gln Trp Leu Met Asn Ser Lys Arg Ser Gly Gly Pro Thr.

The term OXM used in this text also covers any analogue of the above OXMsequence, wherein the histidine residue at position 1 is maintained orreplaced by an aromatic moiety carrying a positive charge or aderivative thereof, preferably wherein the moiety is an amino acid, morepreferably wherein it is a histidine derivative, while 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of theother amino acids in the above OXM sequence can be independentlyreplaced by any other independently chosen amino acid, with theexception of histidine in position 1.

Any one or more (to 22) other alpha-amino acid residue in the sequencecan be independently replaced by any other one alpha-amino acid residue.Preferably, any amino acid residue other than histidine is replaced witha conservative replacement as well known in the art i.e. replacing anamino acid with one of a similar chemical type such as replacing onehydrophobic amino acid with another.

As discussed above, 1 to 22 of the amino acids can be replaced. Inaddition to the replacement option above, this may be by a non-essentialor modified or isomeric form of an amino acid. For example, 1 to 22amino acids can be replaced by an isomeric form (for example a D-aminoacid), or a modified amino acid, for example a nor-amino acid (such asnorleucine or norvaline) or a non-essential amino acid (such astaurine). Furthermore, 1 to 22 amino acids may be replaced by acorresponding or different amino acid linked via its side chain (forexample gamma-linked glutamic acid). For each of the replacementsdiscussed above, the histidine residue at position 1 is unaltered ordefined above.

In addition, 1, 2, 3, 4 or 5 of the amino acid residues can be removedfrom the OXM sequence with the exception of histidine at the 1 position(or as defined above). The deleted residues may be any 2, 3, 4 or 5contiguous residues or entirely separate residues.

The C-terminus of the OXM sequence may be modified to add further aminoacid residues or other moieties. The OXM above may be provided as thecorresponding salt thereof. Examples of pharmaceutically acceptablesalts of OXM and its analogues include those derived from organic acidssuch as methanesulphonic acid, benzenesulphonic acid andp-toluenesulphonic acid, mineral acids such as hydrochloric andsulphuric acid and the like, giving methanesulphonate,benzenesulphonate, p-toluenesulphonate, hydrochloride and sulphate, andthe like, respectively or those derived from bases such as organic andinorganic bases. Examples of suitable inorganic bases for the formationof salts of compounds for this invention include the hydroxides,carbonates, and bicarbonates of ammonia, lithium, sodium, calcium,potassium, aluminium, iron, magnesium, zinc and the like. Salts can alsobe formed with suitable organic bases. Such bases suitable for theformation of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form salts. Such organic bases are alreadywell known in the art and may include amino acids such as arginine andlysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, andtriethanolamine, choline, mono-, di-, and trialkylamines, such asmethylamine, dimethylamine, and trimethylamine, guanidine;N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine;N-benzylphenethylamine; tris(hydroxymethyl)aminomethane; and the like.

Salts may be prepared in a conventional manner using methods well knownin the art. Acid addition salts of said basic compounds may be preparedby dissolving the free base compounds in aqueous or aqueous alcoholsolution or other suitable solvents containing the required acid. WhereOXM contains an acidic function a base salt of said compound may beprepared by reacting said compound with a suitable base. The acid orbase salt may separate directly or can be obtained by concentrating thesolution eg. by evaporation. OXM may also exist in solvated or hydratedforms.

The OXM of the present invention may be conjugated to one or more groupssuch as a lipid, sugar, protein or polypeptide. The OXM can beconjugated by being attached to the group (for example via a covalent orionic bond) or can be associated therewith. The conjugated link ispreferably not through the C or N terminus amino acid, when the OXM isattached to the group. The OXM can be conjugated to a polymer such aspolyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol,polyoxyethylene-polyoxypropylene copolymers, polysaccharides such ascellulose, cellulose derivatives, chitosan, acacia gum, karaya gum, guargum, xanthan gum, tragacanth, alginic acid, carrageenan, agarose, andfurcellarans, dextran, starch, starch derivatives, hyaluronic acid,polyesters, polyamides, polyanhydrides, and polyortho esters.

The OXM can be chemically modified. In particular, the amino acid sidechains, the N terminus and/or the C acid terminus of OXM can bemodified. For example, the OXM can undergo one or more of alkylation,disulphide formation, metal complexation, acylation, esterification,amidation, nitration, treatment with acid, treatment with base,oxidation or reduction. Methods for carrying out these processes arewell known in the art. In particular the OXM is provided as a loweralkyl ester, a lower alkyl amide, a lower dialkyl amide, an acidaddition salt, a carboxylate salt or an alkali addition salt thereof. Inparticular, the amino or carboxylic termini of the OXM may bederivatised by for example, esterification, amidation, acylation,oxidation or reduction. In particular, the carboxylic terminus of theOXM can be derivatised to form an amide moiety.

The OXM can be treated with metals, in particular with divalent metals.For the purposes of this invention the OXM can therefore be provided inthe presence of one or more of the following metals, zinc, calcium,magnesium, copper, manganese, cobalt, molybdenum or iron.

The OXM can be provided in combination with a pharmaceuticallyacceptable carrier or diluent. Suitable carriers and/or diluents arewell known in the art and include pharmaceutical grade starch, mannitol,lactose, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil,alcohol, detergents, emulsifiers or water (preferably sterile). Thecomposition may be a mixed preparation of a composition or may be acombined preparation for simultaneous, separate or sequential use(including administration). The OXM can be provided as a crystallinesolid, a powder, an aqueous solution, a suspension or in oil.

The compositions according to the invention for use in theaforementioned indications may be administered by any convenient method,for example by oral (including by inhalation), parenteral, mucosal (e.g.buccal, sublingual, nasal), rectal, subcutaneous or transdermaladministration and the compositions adapted accordingly.

For oral administration, the composition can be formulated as liquids orsolids, for example solutions, syrups, suspensions or emulsions,tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or physiologically acceptable salt in a suitable aqueousor non-aqueous liquid carrier(s) for example water, ethanol, glycerine,polyethylene glycol or an oil. The formulation may also contain asuspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, powders, granules or pelletscontaining the active ingredient can be prepared using standard carriersand then filled into a hard gelatin capsule; alternatively, a dispersionor suspension can be prepared using any suitable pharmaceuticalcarrier(s), for example aqueous gums, celluloses, silicates or oils andthe dispersion or suspension then filled into a soft gelatin capsule.

Compositions for oral administration may be designed to protect theactive ingredient against degradation as it passes through thealimentary tract, for example by an outer coating of the formulation ona tablet or capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or physiologically acceptable salt in a sterile aqueous ornon-aqueous carrier or parenterally acceptable oil, for examplepolyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil orsesame oil. Alternatively, the solution can be lyophilised and thenreconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently beformulated as aerosols, drops, gels and powders. Aerosol formulationstypically comprise a solution or fine suspension of the active substancein a physiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomising device. Alternatively the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal once the contents of the container have been exhausted. Wherethe dosage form comprises an aerosol dispenser, it will contain apharmaceutically acceptable propellant. The aerosol dosage forms canalso take the form of a pump-atomiser.

Compositions suitable for buccal or sublingual administration includetablets, lozenges and pastilles, wherein the active ingredient isformulated with a carrier such as sugar and acacia, tragacanth, orgelatin and glycerin.

Compositions for rectal or vaginal administration are conveniently inthe form of suppositories (containing a conventional suppository basesuch as cocoa butter), pessaries, vaginal tabs, foams or enemas.

Compositions suitable for transdermal administration include ointments,gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet,capsule or ampoule.

The OXM can be used as a prophylaxis to prevent excess weight gain orcan be used as a therapeutic to lose excess weight.

The excess weight is typically obesity, although the mammal will not becertified as clinically obese in order to be suffering from excessweight. The OXM may be in liquid, solid or semi-solid form.

In today's society, the prevention or treatment of excess weight in amammal is a real need. Preferably the mammal is a human, although it mayalso include other mammalian animals, such as horses, canine animals (inparticular domestic canine animals), feline animals (in particulardomestic feline animals) as well as mammals which are produced for meat,such as porcine, bovine and ovine animals. The present invention can beused to prevent excess weight in such animals in order to maximise leanmeat production.

Throughout this text, the term “prevention” means any effect whichmitigates any excess weight, to any extent. Throughout this text, theterm “treatment” means amelioration of excess weight, to any extent.

According to a second aspect, the present invention provides a methodfor the prevention or treatment of excess weight in a mammal, the methodcomprising administering a composition comprising OXM to a mammal. Themammal is likely to be in need of prevention or treatment of excessweight. The weight loss may be cosmetic. The composition comprising OXMwill be administered in an effective concentration.

All preferred features of the first aspect of the invention, also applyto the second.

A third aspect of the present invention provides a method for cosmeticweight loss in a mammal, the method comprising administering acomposition comprising OXM to a mammal. In this circumstance, the weightloss is purely for the purposes of cosmetic appearance.

All preferred features of the first and second aspects also apply to thethird.

Without being bound to this theory, it is understood that the presentinvention provides the prevention or treatment of excess weight by theadministration of OXM which acts as an inhibitor to food intake to themammalian body. Such reduced food intake results in the prevention ortreatment of excess weight in a mammal. In this text the term “food”includes a substance which is ingested and which has calorific value.

A fourth aspect to the present invention provides the use of OXM in themanufacture of a medicament for the prevention or treatment of excessweight.

All preferred features of the first and third aspects, all apply to thefourth.

The first, second and fourth aspects of the invention relate tomedicaments, the particular dosage regime for which will ultimately bedetermined by the attending physician and will take into considerationsuch factors as the OXM being used, animal type, age, weight, severityof symptoms and/or severity of treatment to be applied, method ofadministration of the medicament, adverse reaction and/or contraindications. Specific defined dosage ranges can be determined bystandard designed clinical trials with patient progress and recoverybeing fully monitored.

Such trials may use an escalating dose design using a low percentage ofthe maximum tolerated dose in animals as the starting dose in man.

The fifth aspect of the present invention relates to the use of OXM toidentify an agent which inhibits food intake in a mammal. This aspect ofthe invention provides a means to identify and develop further suitablemedicaments for the prevention or treatment of excess weight.

The use of OXM may include the use of the peptide itself or may includethe use of the theoretical or models characteristics of OXM. Thefunctional or structural characteristics of OXM utilised may be of apeptide itself or may be of a computer-generated model, a physical twoor three-dimensional model or an electrical generated (e.g. computergenerated) primary, secondary or tertiary structure, as well as thepharmacophore (three-dimensional electron density map) or its X-raycrystal structure.

The structural characteristics may enable the identification ofpotential agents that may interact with OXM thereby affecting it'sfunction. The identification may be by computer modelling and/orrational drug design.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis.

The present invention is now described by way of example only and withreference to the following figures, in which:

FIG. A is a graphical representation of preproglucagon and its componentparts;

FIG. 1 is a comparison of the effects of ICV and iPVNproglucagon-derived and related products on food intake in fasted rats.FIG. 1A illustrates the cumulative food intake (g) up to 8 h after ICVinjection of GLP-1, OXM, glucagon, or glicentin (all 3 nmol) into fastedanimals. *, P<0.05 vs. saline control. FIG. 1B illustrates cumulativefood intake (g) up to 24 h after an acute iPVN injection of GLP-1, OXM(both 1 nmol), or exendin-4 (0.03 nmol) into fasted animals. *, P<0.01vs. saline control for all groups at 1, 2, and 4 h. *, P<0.05 vs. salinecontrol for exendin-4 only at 8 h;

FIG. 2 shows two graphs of the effects of ICV and iPVN OXM on foodintake in fasted rats. FIG. 2A, cumulative food intake (g) up to 8 hafter an acute ICV injection of OXM (0.3, 1, 3, or 10 nmol). FIG. 2B,cumulative food intake (g) up to 8 h after an acute iPVN injection ofOXM (0.1, 0.3, or 1.0 nmol) into fasted animals. *, P<0.05 vs. salinecontrol;

FIG. 3 shows two bar graphs of the effect of ICV OXM at the onset of thedark phase. Sated rats received an ICV injection of OXM, GLP-1 (3 nmol),or saline at the onset of the dark phase. Food intake (grams; A) andbehaviors (B) at 1 h postinjection were determined. *, P<0/05 vs. salinecontrol;

FIG. 4 shows two bar graphs of the inhibition of OXM and GLP-1 effectson food intake by exendin-(9-39). FIG. 4A, food intake 1 h after anacute ICV injection of GLP-1 (3 nmol), GLP-1 plus exendin-(9-39) (30nmol), OXM (3 nmol), OXM and exendin-(9-39) (30 nmol), or exendin-(9-39)alone (30 nmol). FIG. 4B, food intake after an acute iPVN injection ofGLP-1 (1 nmol), GLP-1 and exendin-(9-39) (10 nmol), OXM (1 nmol), OXMand exendin-(9-39) (10 nmol), or exendin-(9-39) alone (10 nmol) intofasted animals. **, P<0.005 vs. saline control;

FIG. 5 is a graph of the competition of [¹²⁵I] GLP-1 binding in rathypothalamic membranes by GLP-1 and OXM;

FIG. 6 illustrates the effect of a) IP OXM (30, 100 and 300 nmol/kg in500 μl saline) or saline on cumulative food intake (g) in 24-hour fastedrats injected during the early dark phase (closed squares=saline, opencircles=OXM 30 nmol/kg, closed triangles=OXM 100 nmol/kg, opentriangles=OXM 300 nmol/kg); and b) IP OXM (30 and 100 nmol/kg in 500 μlsaline) or saline on cumulative food intake in non-fasted rats injectedprior to the onset of the dark phase (closed squares=saline, opencircles=OXM 30 nmol/kg, closed triangles=OXM 100 nmol/kg). *P<0.05 vs.saline;

FIG. 7 illustrates the effect of twice daily IP injections of OXM (50nmol/kg) or saline for seven days on a) cumulative food intake (g); andb) body weight gain (g). *P<0.05, **P<0.01, ***P<0.005 vs. saline;

FIG. 8 illustrates the effect of IP OXM (50 nmol/kg), saline or apositive control (1 hour=GLP-1 (50 nmol/kg); 2 hours=CCK (15 nmol/kg))on gastric emptying in 36-hour fasted rats. Contents (dry weight) of thestomach were expressed as a percentage of the food intake during the30-minute feeding period. **P<0.01 vs. saline;

FIG. 9 illustrates the effect of increasing doses of OXM (0.01-1.0nmole) on 1 hour food intake when administered into the arcuate nucleusof 24-hour fasted rats. *P<0.05, **P<0.01, ***P<0.05 vs. saline;

FIG. 10 illustrates the effect of iARC administration of exendin 9-39 (5nmoles) or saline injected 15 minutes prior to IP administration of OXM(30 nmol/kg), GLP-1 (30 nmol/kg) or saline on 1 hour food intake (g).(S=saline, G=GLP-1 (30 nmol/kg), Ox=OXM (30 nmol/kg), Ex=exendin 9-39 (5nmoles));

FIG. 11 a illustrates the expression of fos-like immunoreactivity inresponse to A) IP saline or B) IP OXM (50 nmol/kg) in the arcuatenucleus of the hypothalamus (×40 magnification). ***P<0.005 vs. saline;and

FIG. 11 b illustrates the expression of fos-like immunoreactivity inresponse to A) IP saline, B) IP OXM (50 nmol/kg) or C) IP CCK (15nmol/kg) in the NTS and AP of the brainstem.

EXAMPLES A-OXM Causes a Potent Decrease in Fasting-Induced Refeedingwhen Injected Both ICV and iPVN Peptides and Chemicals

GLP-1, glicentin, glucagon, and SP-1 were purchased from PeninsulaLaboratories, Inc. (St. Helens, UK). OXM was purchased from IAF BioChemPharma (Laval, Canada). Exendin-4 and exendin-(9-39) were synthesised atMedical Research Council, Hemostasis Unit, Clinical Sciences Center,Hammersmith Hospital, London, UK using F-moc chemistry on an 396 MPSpeptide synthesiser (Advanced ChemTech, Inc.) and purified by reversephase HPLC on a C₈ column (Phenomex, Macclesfield, UK). The correctmolecular weight was confirmed by mass spectrometry. All chemicals werepurchases from Merck & Co. (Lutterworth, Leicester, UK) unless otherwisestated.

Animals

Adult male Wistar rats (ICSM, Hammersmith Hospital) were maintained inindividual cages under controlled conditions of temperature (21-23° C.)and light (12 h of light, 12 h of darkness) with ad libitum access tofood (RM1 diet, Special Diet Services UK Ltd., Witham, UK) and tapwater. Animals were handled daily after recovery from surgery untilcompletion of the studies. All animal procedures undertaken wereapproved by the British Home Office Animals (Scientific Procedures Act1986 (Project License PIL 90/1077).

ICV and iPVN Cannulation and Infusions of Test Compounds

Animals had permanent stainless steel guide cannulas (Plastics One,Roanoke, Va.) stereotactically implanted ICV or iPVN. All studies werecarried out in the early light phase, between 0900-1100 h, after a 24-hfast, and food intake was measured 1, 2, 4, 8, and 24 h postinjection.

Feeding Study Protocols

Comparison of the effect of proglucagon-derived products and relatedpeptides on food intake.

In study 1a, rats were injected ICV with 10 μl saline, GLP-1 (13 nmol),OXM (3 nmol), glucagon (3 nmol), or glicentin (3 nmol; n=8/group).

In all studies, the OXM with the following sequence was used:

(SEQ ID NO: 1) His Ser Gln Gly Thr Phe Thr Ser Asp TyrSer Lys Tyr Leu Asp Ser Arg Arg Ala GlnAsp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn Lys Asn Asn Ile Ala.

Human GLP-1 with the following sequence was used:

(SEQ ID NO: 5) His Ala Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg.

In study 1b, rats were injected iPVN with 1μ saline, GLP-1 (1.0 nmol),OXM (1.0 nmol), glicentin (1.0 nmol), glucagon (1.0 nmol), or SP-1 (3.0nmol; n=12-15/group). Exendin-4, when injected ICV, inhibits food intakemore potently than GLP-1. Therefore, exendin-4 was injected iPVN at adose of 0.03 nmol.

Investigation of the Effect of Increasing Doses of OXM on Food Intake

In study 2a, rats were injected ICV with saline, GLP-1 (3 nmol), or OXM(0.3, 1, 3 or 10 nmol; n=8/group). In study 2b, rats were injected iPVNwith saline, GLP-1 (1.0 nmol), or OXM (0.1, 0.3, or 1.0 nmol;n−12-15/group). To assess whether OXM acts via the GLP-1 receptor, astudy using the GLP-1 receptor antagonist exendin-(9-39) was performed.

Night Time Feeding and Behavioural Analysis.

Study 3. It is possible that OXM inhibits food intake via nonspecifictaste aversion, and that it is not a true satiety factor. Therefore, ICVcannulated rats were administered GLP-1 (3 nmol), OXM (3 nmol), orsaline (n=6/group) at the onset of the dark phase. Food intake wasmeasured 1 h postinjection (study 3a), and behaviour was assessed (study3b). Rats were observed for 1 h postinjection using a behavioural scoresheet.

In study 4a, rats were injected with ICV with saline, GLP-1 (3 nmol),GLP-1 (3 nmol) plus exendin-(9-39) (30 nmol), OXM (3 nmol), OXM (3 nmol)plus exendin-(9-39) (30 nmol), or exendin-(9-39) alone (30 nmol). Instudy 4b, rats were iPVN injected with saline, GLP-1 (1 nmol), GLP-1 (1nmol) plus exendin-(9-39) (10 nmol), OXM (1 nmol), OXM (1 nmol) plusexendin-(9-39) (10 nmol), or exendin-(9-39) alone (10 nmol;n=10-12/group).

Receptor Binding Assays. Study 5.

Receptor binding assays were performed in a final volume of 0.5 ml rathypothalamic membranes (200μg protein), 500 Bq (100 pM) [¹²⁵I]GLP-1, andunlabeled competing peptides (GLP-1 and OXM) as specified. Membraneswere incubated at room temperature for 90 min. Bound and freeradioactivity were separated by centrifugation (2 min, 4° C.). Pelletedmembranes were washed with assay buffer (0.5 ml, ice-cold), and themembranes were centrifuged as described above. The supernatant wasremoved, and the radioactivity in the pellet was counted using aγ-counter. Specific (saturable) binding was calculated as the differencebetween the amount of [¹²⁵I]GLP-1 bound in the absence (total binding)and presence of 1 μm GLP-1 or OXM (nonsaturable binding). All curveswere constructed with points in triplicate. IC₅₀ values were calculatedusing the Prism 3 program (GraphPad Software, Inc., San Diego, Calif.).

Statistics

For food intake analyses, data are presented as the mean±SEM.Statistical differences between experimental groups were determined byANOVA, followed by a post-hoc least significant difference test (Systat8.0, Evanston, Ill.). For behavioural analyses, data are expressed asthe median number of occurrences of each behaviour and the range.Comparisons between groups were made using the Mann-Whitney U test(Systat 8.0). In all cases, P<0.05 was considered statisticallysignificant.

Results

Comparison of the effects of proglucagon-derived products and relatedpeptides on food intake

ICV Administration.

In study 1a, OXM and GLP-1 (3 nmol) significantly reduced refeeding.This inhibition of food intake lasted until 4 h postinjection (FIG. 1A).Glucagon and glicentin (3 nmol) failed to affect food intake at any timepoint (FIG. 1A).

iPVN Administration.

In study 1b, OXM, GLP-1 (3 nmol) and exendin-4 (0.03 nmol) alsoinhibited refeeding when injected iPVN. This inhibition lasted at least8 h postinjection, longer than when injected ICV (FIG. 1B). Glicentin,glucagon (1 nmol), and SP-1 (3 nmol) failed to affect food intake at anytime point when injected iPVN.

Effects of Increasing Doses of OXM on Food Intake ICV Administration.

In study 2a, when injected ICV, OXM reduced refeeding in adose-dependent manner, reaching a maximal effect at a dose of 3 nmol 1,2, and 4 h postinjection (FIG. 2A).

iPVN Administration.

In study 2b, food intake was significantly reduced by iPVN-injectedGLP-1 and OXM (both 1 nmol) until 8 h postinjection (FIG. 2B).

Effect of OXM in ICV-Cannulated Sated Rats at the Onset of the DarkPhase.

The dark phase is the rats' natural feeding time. Therefore, assessingthe effect of a putative satiety factor in non-fasted animals at thistime would represent a more physiological effect.

Effect of OXM on Food Intake.

In study 3a, when injected in the early dark phase, both GLP-1 and OXM(3 nmol) significantly reduced food intake compared with that ofsaline-treated animals 1 h postinjection [FIG. 3A].

Observation of Behaviour after ICV Injection of OXM.

ICV administration of OXM (3 nmol) in the early dark phase led to asignificant decrease in feeding episodes (study 3a) and an increase inrearing behaviour (study 3b) [FIG. 3B]. There was no change in grooming,still, head down, burrowing, or locomotion episodes.

To assess whether OXM acts via the GLP-1R, a study using the GLP-1Rantagonist, exendin-(9-39) was performed.

ICV Administration. Study 4.

ICV coadministration of the GLP-1 receptor antagonist exendin-(9-39)with GLP-1 at a ratio of 10:1 (antagonist/agonist) blocked the anorecticeffects of GLP-1 [FIG. 4A]. Furthermore, coadministration ofexendin-(9-39) with OXM resulted in attenuation of the anorectic effectof OXM [FIG. 4A].

iPVN Administration.

Similarly, when injected iPVN, the anorectic effects of both GLP-1 andOXM were blocked when coinjected with exendin-(9-39) [FIG. 4B].

Receptor Binding Assays. Study 5.

The affinity (IC₅₀) of GLP-1 for the GLP-receptor in rat hypothalamicmembrane preparations was 0.16 nM (FIG. 5). The affinity of OXM for theGLP-1 receptor in the same membrane preparations was 8.2 nM (FIG. 5),which is approximately 2 orders of magnitude weaker than that of GLP-1.

Discussion.

OXM causes a potent decrease in fasting-induced refeeding when injectedboth ICV and iPVN. The effect was sustained until 8 h (iPVN) or 4 h(ICV) postinjection. The effect of OXM is approximately of the samemagnitude and time course as that of GLP-1 when administered ICV andiPVN at equimolar doses. In addition, OXM inhibits food intake innonfasted rats at the onset of the dark phase, and at that time theyshowed no signs of aversive behaviour.

It has been suggested that there is an OXM-specific binding site ingastric mucosa. However, no such binding site has been identified in theCNS. Therefore, it was proposed that OXM mediated its effects via thehypothalamic GLP-IR, as GLP-1 and OXM have similar potency in feedingstudies. It has been shown that OXM has a nanomolar affinity for theGLP-IR (IC₅₀=8.2 nM). This affinity is approximately 2 orders ofmagnitude weaker than that of GLP-1 (IC₅₀=0.16 nM). Yet despite thisreduced affinity for the GLP-1R, OXM reduces food intake to the samemagnitude. One explanation for this is that OXM might act through boththe GLP-1R and its own receptor in the hypothalamus. Thus, OXM couldelicit a response comparable to that of GLP-1 despite its lower affinityfor the GLP-IR.

Exendin-(9-39), a fragment of the GLP-1R agonist exendin-4, is a potentand selective antagonist at the GLP-1R. When GLP-1 and exendin-(9-39)are coinjected, the anorectic actions of GLP-1 are blocked. When OXM iscoinjected with exendin-(9-39), the anorectic effects of OXM are alsocompletely blocked. This would strengthen the argument that OXM ismediating its effects via the GLP-1R.

We investigated the effects of glicentin, and glucagon after an acuteICV injection in fasted rats. No effect on fasting-induced food intakewas seen after the administration of these peptides. In addition, therewas no effect of these peptides when they were administered iPVN. WhenSP-1, the putative minimal active structure of OXM, was injected iPVN,no inhibition of food intake was observed. Therefore the effect seen byOXM is specific.

B-Peripheral Administration of OXM Also Reduces Food Intake and BodyWeight Gain Peptides and Chemicals

OXM was purchased from IAF BioChem Pharma (Laval, Canada). GLP-1 waspurchased from Peninsula Laboratories Inc. (St. Helens, UK). Exendin9-39 was synthesised at Medical Research Council, Hemostasis Unit,Clinical Sciences Centre, Hammersmith Hospital, London, UK using F-mocchemistry on a 396 MPS peptide synthesizer (Advanced ChemTech Inc.,Louisville, Ky.) and purified by reverse phase HPLC on a C₈ column(Phenomex, Macclesfield, UK), using a gradient of acetonitrile on 0.1%trifluoroacetic acid. Correct molecular weight was confirmed by massspectrometry. All chemicals were purchases from Merck Eurolab Ltd.(Lutterworth, Leicestershire, UK), unless otherwise stated.

Animals

Adult male Wistar rats (180-200 g) were maintained in individual cagesunder controlled conditions of temperature (21-23° C.) and light (12hours light, 12 hours dark) with ad libitum access to standard rat chow(RM1 diet, Special Diet Services UK Ltd., Witham, Essex, UK) and water.All procedures undertaken were approved by the British Home OfficeAnimals (Scientific Procedures) Act 1986 (Project Licenses PPL: 90/1077,70/5281 and 70/5516).

Intra-Arcuate Nucleus Cannulation

Animals had permanent indwelling, unilateral, stainless steel guidecannulae (Plastics One, Roanoke, Va.) stereotactically implanted intothe arcuate nucleus of the hypothalamus, using a cannulation protocolusing cannulae positioned 3.3 mm posterior to and 0.3 mm lateral tobregma and 9.0 mm below the outer surface of the skull.

Intra-Peritoneal (IP) Injections

All IP injections were delivered using a 1 ml syringe and a 25 gaugeneedle. The maximum volume of injection was 500 μA and was adjustedaccording the weight of the individual animal. All peptides weredissolved in saline.

In these studies, the human OXM and human GLP-1 were used with thesequences provided on pages 15 and 16 above.

In Vivo Protocols 1. Investigating the Dose-Response Effect ofPeripheral Administration of OXM on Food Intake in Fasted Animals:

Animals were fasted for 24 hours prior to the study. During the earlylight phase (09.00-10.00 hr), rats were given a single IP injection ofsaline, GLP-1 (30 nmol/kg body weight as a positive control) or OXM(10-300 nmol/kg body weight) (n=12 per group) in a volume of 500 μl.Following the injection, the animals were returned to their home cagesand provided with a pre-weighed amount of chow. Food intake was measured1, 2, 4, 8 and 24 hours post-injection.

2. Investigating the Effect of Peripheral Administration of OXM on FoodIntake in Non-Fasted Animals During the Dark Phase:

The dark phase is the “normal” feeding time for rats. Therefore, anyinhibition of food intake at this time could be considered to be morephysiological than alterations to refeeding following a fast. Animalsreceived a single IP injection of saline or OXM (3-100 nmol/kg bodyweight) (n=12 per group) prior to lights out (18.00-19.00 hr). Foodintake was measured 1, 2, 4, 8 and 12 hours post-lights-out.

3. The Effect of Repeated IP Injections of OXM

45 animals were randomised by weight into three groups (n=15 pergroup): 1) Saline-treated with ad libitum access to food, 2) OXM-treated(50 nmol/kg body weight per injection—a dose based on the previousdose-response experiment) with ad libitum access to food, 3)Saline-treated, but food restricted to the mean light and dark phasefood intake of the OXM-treated group. Animals were injected twice daily(07.00 and 18.00 hr) for seven days. Food intake (g), body weight (g)and water intake (ml) were measured daily. On the eighth day, theanimals were killed by decapitation. Epididymal white adipose tissue(WAT) and interscapular brown adipose tissue (BAT) were removed andweighed as an assessment of body adiposity.

4. Investigating the Effect of Peripheral Administration of OXM onGastric Emptying

Animals were fasted for 36 hours to ensure that the stomach was empty.During the early light phase (09:00-10:00) were allowed ad libitumaccess to a pre-weighed amount of standard rat chow for thirty minutes.After that time, the food was removed and reweighed. The animals werethen IP injected with saline, OXM (50 nmol/kg body weight) or CCK-8 (15nmol/kg body weight). Rats were then killed at the same times as thoseused in the previous feeding studies: 1, 2, 4 or 8 hours post-feeding(n=12 per group per time-point). The CCK-8 group was used as a positivecontrol for the experiment at the two-hour time-point only. Animals werekilled by carbon dioxide asphyxiation. A laparotomy was rapidlyperformed and the stomach exposed. The pyloric junction was ligated (2.0Mersilk, Johnson & Johnson, Belgium), followed by ligation of thegastro-oesophogeal junction and the stomach was removed. The gastriccontents were then removed, placed in a pre-weighed weighing boat andleft to air-dry for 48 hours. Once dry, the contents were weighed andthe percentage of the chow ingested during the half-hour re-feedingperiod remaining in the stomach per rat was then calculated using thefollowing formula:

${\% \mspace{14mu} {food}\mspace{14mu} {remaining}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {stomach}} = {\frac{\begin{matrix}{{{dry}\mspace{14mu} {weight}\mspace{14mu} {of}}\mspace{14mu}} \\{{stomach}\mspace{14mu} {content}}\end{matrix}}{{weight}\mspace{14mu} {of}\mspace{14mu} {food}\mspace{14mu} {ingested}} \times 100}$

5. Investigating the Effect of Increasing Doses of Intra-Arcuate OXM

Intra-arcuate (Intra-ARC (iARC)) cannulated rats (n=12-15 per group)were randomised by weight into 6 groups. During the early light phase(0900-1000), 24-hour fasted rats received an iARC injection of saline,OXM (0.01, 0.03, 0.1, 0.3 or 1.0 nmoles). Food intake was measured 1, 2,4, 8 and 24 hours post-injection.

6. Investigating Whether Peripherally Administered OXM is ActingDirectly Via Arcuate Nucleus GLP-1 Receptors.

Rats cannulated into the arcuate nucleus were randomised into 6 groups(n=10-12 per group). During the early light phase (0900-1000) 24-hourfasted rats received an iARC injection of saline or exendin₉₋₃₉ (5nmoles) followed by an IP injection of saline, OXM (30 nmoles/kg bodyweight) or GLP-1 (30 nmoles/kg body weight) 15 minutes later. Theinjection details are described in Table 1 below.

TABLE 1 Intra-ARC Group injection IP injection 1 Saline Saline 2 SalineOXM (30 nmoles/kg) 3 Saline GLP-1 (30 nmoles/kg) 4 Exendin 9-39 Saline(5 nmoles) 5 Exendin 9-39 OXM (30 nmoles/kg) (5 nmoles) 6 Exendin 9-39GLP-1 (30 nmoles/kg) (5 nmoles)

Immunohistochemistry

90 minutes after an IP injection of OXM (50 nmol/kg), CCK (15 nmol/kg)or saline, rats were terminally anaesthetized was transcardiallyperfused with 0.1 M phosphate buffered saline (PBS) following by 4%PB-formalin (PBF). The brains were removed and post-fixed overnight inPBF and then transferred to PB-sucrose (20% w/v) overnight. 40 μmcoronal sections of brain and brainstem were cut on a freezing microtomeand stained for fos-like immunoreactivity (FLI) by theavitin-biotin-peroxidase method. The sections were then mounted onpoly-L-lysine-coated slides, dehydrated in increasing concentrations ofethanol (50-100%), delipidated in xylene and coverslipped using DPXmountant. Slides were examined for FLI-positive nuclei using a lightmicroscope (Nikon Eclipse E-800) and images captured using a microimager(Xillix MicroImager). The numbers of FLI-positive nuclei in thehypothalamus and brainstem were counted by an independent member of theresearch team who was blinded to the experimental groups. The averagenumber of FLI-positive nuclei per section was calculated and expressedas an integer for each animal.

Hypothalamic Explant Static Incubation

A static incubation system was. Male Wistar rats were killed bydecapitation and the whole brain removed immediately. The brain wasmounted, ventral surface uppermost, and placed in a vibrating microtome(Microfield Scientific Ltd., Dartmouth, UK). A 1.7 mm slice was takenfrom the basal hypothalamus, blocked lateral to the Circle of Willis andincubated in chambers containing 1 ml of artificial cerebrospinal fluidwhich was equilibrated with 95% O₂ and 5% CO₂. The hypothalamic sliceencompassed the medial pre-optic area, PVN (paraventricular hypothalamicnucleus), dorsomedial nucleus, ventromedial nucleus, lateralhypothalamus and ARC. The tubes were placed on a platform in a waterbath maintained at 37 C. After an initial 2-hour equilibration period,each explant was incubated for 45 minutes in 600 μl aCSF (basal period)before being challenged with a test period. OXM, 100 nM was used as adose representing a concentration ten times that of its IC₅₀ for theGLP-1 receptor. The viability of the tissue was confirmed by a final45-minute exposure to aCSF containing 56 mM KCl. At the end of eachexperimental period, the aCSF was removed and stored at −20° C. untilmeasurement of αMSH-immunoreactivity by radioimmunoassay.

Radioimmunassay to Measure αMSH-IR

Alpha-MSH was measured using an in-house radioimmunoassay, developedusing an antibody from Chemicon International Inc.

Statistical Analysis

Data from IP and iARC feeding studies were analyzed by ANOVA withpost-hoc LSD (least significant difference) test. Fat pad weights fromdifferent treatment groups were analyzed using an unpaired t test. Datafrom the hypothalamic explant incubation study, in which each explantwas compared with its own basal period, were analyzed by paired t test.In all cases P<0.05 was considered to be statistically significant.

Results 1. The Effect of Peripheral Administration of OXM in FastedAnimals:

Intraperitoneal administration of OXM (100 nmol/kg and 300 nmol/kg)caused a significant inhibition in refeeding in 24-hour fasted animalsone hour post-injection, compared with saline controls (1 hour: OXM 100nmol/kg, 5.4±0.2 g (P<0.05), 300 nmol/kg, 4.5±0.2 g (P<0.05) vs. saline,6.3±0.2 g). The reduction in food intake caused by 100 nmol/kg wassustained until 8 hours post-injection. However, the highest dose of OXM(300 nmol/kg) continued to significantly inhibited food intake 24 hourspost-injection (24 hours: OXM, 300 nmol/kg, 9.5±0.6 g vs. saline,17.5±0.7 g; P<0.05) (FIG. 6 a). The 30 nmol/kg and 10 nmol/kg failed toalter food intake at any time-point investigated.

2. The Effect of Peripheral Administration of OXM in Non-Fasted Animalson Dark Phase Food Intake:

OXM, 3 and 10 nmol/kg, failed to affect food intake at any time-pointinvestigated in nocturnally feeding rats injected immediately prior tothe dark phase. However, OXM, 30 nmol/kg, significantly inhibited foodintake until 2 hours post-injection (2 hours: OXM, 30 nmol/kg, 4.5±0.4 gvs. saline, 5.8±0.4 g; P<0.05). Food intake was reduced 4 hourspost-injection, but this was not significant. OXM, 100 nmol/kg,significantly inhibited food intake throughout the dark phase (8 hours:OXM, 100 nmol/kg, 14.1±0.8 g vs. saline, 16.9±0.5 g; P<0.05) (FIG. 6 b).

3. The Effect of Repeated IP Administration of OXM

Twice-daily IP injections of OXM (50 nmol/kg) for seven days caused asignificant decrease in cumulative daily food intake, compare withsaline-treated control animals (Cumulative food intake day 7: OXM, 50nmol/kg, 168±4.6 g vs. saline, 180±4.3 g; P<0.01) (FIG. 7 a).Furthermore, OXM-treated animals gained weight significantly more slowlythan saline controls (cumulative weight gain day 7: OXM, 50 nmol/kg,21.0±1.5 g vs. saline, 37.6±1.9 g; P<0.005). Moreover, the foodrestricted “pair fed” animals did not gain weight as slowly asOXM-treated animals, despite receiving the same food intake (Day 7: pairfed, 33.5±2.0 g; P═NS vs. saline (ad libitum fed), P<0.05 vs. OXM) (FIG.7 b). In addition, chronic OXM caused a decrease in adiposity that wasnot seen in saline-injected pair fed animals (Table 2). Water intake wassignificantly reduced in OXM-treated animals on days 1 and 2 of theexperiment (Day 1: OXM, 24.1±1.28 ml vs. saline, 28.1±1.33 ml; P<0.05).On subsequent days, there was an increase in daily water intake comparedwith saline-treated animals (days 3-6). However, by day 7, there was nodifference in water intake between saline and OXM-treated groups (notshown).

TABLE 2 The effect of twice-daily IP administration of saline or OXM (50nmol/kg) for seven days on the weight of epididymal WAT andinterscapular BAT in food restricted and ad libitum fed rats. Tissue/hormone Saline OXM Pairfed WAT 0.69 ± 0.02 0.51 ± 0.01^(a) 0.61 ±0.02^(b) BAT 0.16 ± 0.01 0.12 ± 0.01^(a) 0.15 ± 0.01^(b)

4. The Role of Delayed Gastric Emptying on the Anorectic Effect of OXM:

One hour after food was presented to the 36-hour fasted rats, the dryweight of the contents of the stomachs (as a percentage of the foodconsumed during the 30 minute feeding period) of GLP-1-treated animalswere significantly greater than that of saline-treated animals (1 hour:GLP-1, 50 nmol/kg, 76.9±2.7 g vs. saline, 65.8±1.6 g; P<0.01),suggesting that GLP-1 caused a significant decrease in gastric emptying.The contents of the stomachs of OXM-treated animals were greater thanthose of the saline treated controls, although this was notstatistically significant (1 hour: OXM, 50 nmol/kg, 72.0±1.4 g vs.saline 65.8±1.6 g; P=0.07). Two hours post-feed, OXM did not affect thecontents of the stomach, compared with saline-treated animals. However,animals injected with the positive control for this time-point, CCK (15nmol/kg), had significantly greater stomach content (2 hours: CCK, 15nmol/kg, 64.7±6.4 g vs. saline, 38.5 g; P<0.01), suggesting that CCKcaused a significant decrease in the rate of gastric emptying. There wasno effect of OXM on the contents of the stomach, compared withsaline-treated animals, at 4 or 8 hours post-feed (FIG. 8).

5. Investigating the Effect of Increasing Doses of OXM InjectedIntra-Arcuate Nucleus

Food intake was significantly inhibited by all doses (except 0.01nmoles) of OXM administered iARC during the 1^(st) hour of re-feedingfollowing a 24-hour fast (1 hour: OXM 0.03 nmoles, 6.1±0.5 g (P<0.05);0.1 nmoles, 5.6±0.4 g (P<0.05); 0.3 nmoles, 5.1±0.6 g (P<0.01); 1.0nmole, 3.6±0.5 g (P<0.005) all vs. saline, 7.7±0.2 g) (FIG. 9). OXM 0.3and 1.0 nmoles continued to significantly inhibit food intake until 8hours post-injection. Twenty-four hours post-injection, food intake wasinhibited by OXM 1.0 nmoles, although this was not significant (24hours: OXM, 1.0 nmole, 37.8±3.0 g vs. saline, 40.8±1.6 g; P═NS).

6. Investigating Whether Peripherally Administered OXM is Acting ViaArcuate Nucleus GLP-1 receptors

Intraperitoneal administration of both GLP-1 (30 nmol/kg) and OXM (30nmol/kg) caused a significant inhibition of food intake one hour intothe dark phase (1 hour: GLP-1, 5.0±0.6 g, OXM, 5.1±0.4 g vs. saline,9.2±0.3 g). However, the anorexia caused by IP administration of OXM wasblocked by prior administration of the GLP-1 receptor antagonist,exendin 9-39 (300 nmol/kg), injected directly into the ARC (Table 3 &FIG. 10). Inhibition of food intake by IP GLP-1 was not affected byprior iARC administration of exendin 9-39.

TABLE 3 The effect of iARC administration of exendin 9-39 (5 nmoles) orsaline injected 15 minutes prior to IP administration of OXM (30nmol/kg), GLP-1 (30 nmol/kg) or saline on 1 hour food intake (g). (S =saline, G = GLP-1 (30 nmol/kg), Ox = OXM (30 nmol/kg), Ex = exendin 9-39(5 nmoles)). Peptide Food intake (g) S.E.M. Saline/saline 9.2 0.3Saline/GLP-1 5.0 0.6 Exendin 9-39/GLP-1 5.0 0.3 Saline/OXM 5.1 0.4Exendin 9-39/OXM 9.4 0.4 Exendin 9-39/saline 9.0 0.3

7. Mapping the Expression of FLI in the Hypothalamus in Response IP OXM:

After IP OXM administration (50 nmol/kg) dense staining of FLI was foundalmost exclusively in the hypothalamic arcuate nucleus (FIG. 11 a). Noother hypothalamic nuclei (PVN (paraventricular hypothalamic nucleus),DMH (dorsomedial hypothalamic nucleus), VMH (ventromedial hypothalamicnucleus)) demonstrated specific c-fos staining.

In the brainstem, IP CCK (15 nmol/kg) caused dense staining of FLI, mostnotably in the NTS (nucleus tractus solitarius) and the area postrema(FIG. 6 b). However, neither IP saline nor IP OXM (50 nmol/kg) caused aspecific increase in c-fos expression in the same brainstem nucleiinvestigated (FIG. 11 b).

8. Changes in Alpha-MSH Release from Hypothalamic Explants whenIncubated with OXM

Incubating OXM (100 nM) was hypothalamic explants caused a significantincrease in the release of α-MSH, compared with basal release (α-MSH:OXM, 100 nM, 4.1±0.6 fmol/explant vs. 2.6±0.5 fmol/explant; P<0.005).Explant viability was assessed by incubation with 56 mM KCl, andviability was confirmed in >80% of explants. Those explants that werenot viable were excluded from the analysis.

Discussion

Peripheral administration of OXM causes a reduction in food intake inrats. This was seen following a fast in the light phase and during thenocturnal feeding phase. The anorectic effect was potent and sustainedfor periods up to 24 hours. Twice-daily IP administration of OXM forseven days caused a reduction in daily food intake compared with thosetreated with saline, with no tachyphylaxis. Animals treated with OXMgained significantly less weight than pair fed animals, despite the twogroups receiving identical daily caloric intake. Intraperitonealadministration of OXM did transiently reduce water intake although thiswas not sustained, suggesting that the reduction in the rate of bodyweight gain was not due to dehydration.

On conclusion of the chronic study, epididymal WAT and interscapular BATwere removed and weighed. It was found that there was a reduction in theweights of all fat pads in OXM-treated animals compared with pair-fedanimals, despite identical food intake. Therefore it appears thatperipheral OXM administration is also affecting other metabolicparameters.

A major contributor to satiety is delayed gastric emptying viavagally-mediated mechanism that leads to brainstem activation. BothGLP-1 and OXM are potent inhibitors of gastric emptying in rodents andhumans and in the case of GLP-1, this is thought to be the dominantmechanism through which it promotes satiety. We hypothesized that OXMwas acting in the same way, and that its effects on gastric emptyingwere the cause of sustained anorexia. However, although peripheraladministration of OXM led to a slight delay in gastric emptying in thefirst hour after the re-introduction of food, this was non-significantand the effect was short-lived. This suggested that OXM does slowgastric emptying, but it is not likely to be responsible for the robustand sustained inhibition of food intake.

We report here that peripheral administration of OXM increases FLI inalmost exclusively in the ARC. Furthermore, we found that incubatinghypothalamic explant with OXM caused a significant increase in therelease of the POMC (pro-opiomelanocortin)-derived product, αMSH fromhypothalamic explants. IP OXM did not affect the expression of FLI inthe NTS and AP—areas known to be important in integrating vagallymediated information, further strengthening the notion that OXM is notacting via these pathways.

It is thought that nuclei in the brainstem are the primary site of GLP-1action, and information is subsequently relayed to the hypothalamic PVN,where its anorectic effects are mediated. Direct injection of OXM intothe ARC, even at very low doses caused a robust and sustained inhibitionof food intake, further supporting the hypothesis that that the ARC isthe site of the actions of OXM. Anorectic effects caused by peripheraladministration of OXM were blocked by prior administration ofexendin₉₋₃₉ into the ARC. Interestingly, however, the anorectic actionsof peripherally administered GLP-1 were not. This finding stronglyindicates that OXM is acting via GLP-1 receptors in the ARC. Inaddition, it has identified distinct pathways which mediate the actionsof GLP-1 and OXM.

Taken together, these data demonstrate that OXM is potentially importantin both long and short-term regulation of food intake and body weightmaintenance. Rather than reducing appetite via “traditional” satietypathways, involving slowing of gastric emptying and activation ofbrainstem nuclei, circulating OXM is mediating its anorectic effects viadirect interaction with the ARC, potentially by activating POMC(pro-opiomelanocortin) neurons within the nucleus. Therefore, OXM may beuseful in the treatment or prevention of excess weight such as obesityin mammals, and further represents a novel target for the development oftherapeutic agents in the treatment of excess weight such as obesity inmammals.

1. A composition comprising oxyntomodulin, for use in the prevention ortreatment of excess weight in a mammal.
 2. A composition comprisingoxyntomodulin, for use as claimed in claim 1, wherein the excess weightis obesity.
 3. A composition comprising oxyntomodulin, for use asclaimed in claim 1 or claim 2, in combination with a pharmaceuticallyacceptable excipient.
 4. A composition comprising oxyntomodulin, for useas claimed in any one of claims 1 to 3, in liquid, solid or semi-solidform.
 5. A composition comprising oxyntomodulin, for use as claimed inany one of claims 1 to 4, wherein the mammal is a human.
 6. A method forthe prevention or treatment of excess weight in a mammal, the methodcomprising administering a composition comprising oxyntomodulin to amammal.
 7. A method, as claimed in claim 6, wherein the excess weight isobesity.
 8. A method for cosmetic weight loss in a mammal, the methodcomprising administering a composition comprising oxyntomodulin to amammal.
 9. A method, as claimed in any one of claims 6 to 8, wherein thecomposition comprises oxyntomodulin and a pharmaceutically acceptableexcipient or carrier.
 10. A method, as claimed in any of claims 6 to 9,wherein the weight loss is a result of reduced food intake.
 11. Use ofoxyntomodulin, in the manufacture of a medicament for the prevention ortreatment of excess weight.
 12. The use of oxyntomodulin to identify anagent which inhibits food intake in a mammal.
 13. The use, as claimed inclaim 12, which is of the functional and/or structural characteristicsof oxyntomodulin.
 14. The use, as claimed in claim 12 or claim 13, whichis of an electronically generated model of oxyntomodulin.
 15. The use,as claimed in any one of claims 12 to 14, wherein the characteristic ofoxyntomodulin is its primary, secondary, tertiary structurepharmacophore and/or X-ray crystal structure.
 16. A compositioncomprising oxyntomodulin, for use in the prevention or treatment ofexcess weight in a mammal as hereinbefore described in the examples. 17.A method for the prevention or treatment of excess weight in a mammal,the method as described with reference to the examples.